This disclosure relates to methods of detecting biological material, particularly assays, methods and kits for detecting biowarfare agents such as microorganisms, biological toxins, and the like.
The bioterrorism attacks and hoaxes in the United States in the fall of 2001 placed tremendous burdens on public health and safety organizations charged with the responsibility of testing the thousands of samples that concerned citizens suspect might contain anthrax. With each confirmed case of illness from anthrax reported, thousands of calls were placed to law enforcement and emergency personnel regarding suspicious white powders and the like found in private homes, offices, restaurants, post offices, on subways, and in schools and shopping centers. Overwhelmingly these substances were found to be benign, many as mundane as pizza flour and vanilla pudding mix according to news accounts at the time. One source of many false alarms is cornstarch that is used to sort mass mailings to prevent envelopes from sticking together.
Unfortunately the anthrax scares greatly taxed the resources of health and safety personnel as each substance had to be subjected to expensive and time consuming testing in the field; a laboratory, or both. Among the techniques currently used for testing of samples suspected of containing anthrax are antibody tests, bacterial culture, and DNA testing. Each of these techniques has one or more significant limitations with respect to speed, expense, accuracy, false positives, false negatives, and/or ability to screen for multiple pathogens and toxins in parallel.
One approach currently utilized for field-testing of anthrax and other biowarfare agents are lateral flow test strips. These devices, which function much like home pregnancy tests, utilize antibodies that bind to specific proteins associated with particular pathogens of concern. Since different organisms express different sets of proteins, users of these antibody-based products must stock a different test strip for each of anthrax, ricin, botulinum toxin, SEB (Staphococcal Enterotoxin B), plague, etc. Moreover, since the process of raising antibodies in mammals remains slow and cumbersome, antibody based assays are difficult to manufacture in very large quantities in a short time period that may be required to respond to an unexpected bioterrorist attack.
It would therefore be desirable to have an assay that can be used by first responders to assess substances suspected of being biowarfare agents so as to rapidly and inexpensively eliminate a variety of mundane substances before more advanced testing is employed.
It would also be desirable to have an assay of the aforementioned type that can be used in conjunction with substances suspected of containing a wide range of biowarfare agents.
It would further be desirable to have an assay of the aforementioned type that can be manufactured in large quantities in a short period of time in the event of a surprise terrorist attack.
The present disclosure relates to assays, methods, and kits for testing powders and other samples suspected of containing a biowarfare agent, such as pathogens and/or toxins (such as proteins) secreted thereby. According to certain provided methods, the sample is first collected by a swab or pad or the like, then immersed in or otherwise contacted with one or more reagents that produce a detectable signal (e.g., color) only in the presence of protein. Failure to produce that signal (such as a color change) indicates that the sample likely does not contain biological material, such as a pathogen or toxin. This permits the elimination of a variety of ordinary safe substances such as sugar, dry wall dust, cleaning solutions, etc. from being subjected to further testing. If it is determined that biological material (e.g., protein) likely is present in the sample, the sample may then be subjected to specific tests using antibodies or the like to determine if a particular pathogen or toxin is present.
In addition to testing for protein content, the sample in some embodiments is also tested to determine if it contains sugar, which is a common source of anthrax hoaxes and false alarms. Moreover, the sample in some embodiments is tested for pH to determine if it is either too acidic or too basic to typically sustain life or contain living material.
In one embodiment, reagents are provided in transparent tubes and the sample is collected using swabs. Separate tubes may be provided, for example, to test for protein, sugar, and/or pH.
In another embodiment, pair(s) of pads are employed that are saturated with reagents that, when combined, produce a color in the presence of protein. The sample is collected on one of the pads and sandwiched or pressed against the other pad. If it is determined that protein is present, further analysis of the sample may be performed.
In a specific example, after detection of protein in the sample, one of the pads that has been in contact with the sample is then placed in contact with a stack of membranes under conditions that permit biomolecules to be transferred from the sample to the stack. This transfer process may be carried out generally as described in U.S. patent application Ser. No. 09/718,990 filed Nov. 20, 2000 (incorporated herein by reference in its entirety). A different biomolecule may be identified on each of the membranes (if it is present in the sample), corresponding, for example, to different pathogens or toxic compounds or other biological molecules. Thus, in certain embodiments the user can determine not only that biological material is present in the sample but also which particular pathogens or toxins that are present.
In yet another embodiment, an analytical test strip is employed for the analysis, the test strip having an absorbent carrier impregnated with a protein indicator. The test sample, which in this embodiment usually has been solubilized or dissolved or otherwise comprises a fluid, is brought into contact with the test strip. In those examples where the sample comprises a liquid, the liquid facilitates solubilization of the protein indicator in the absorbent carrier, therefore enabling mixing of the test sample and the indicator and generation of a detectable color change or other signal in the presence of protein. The test strip may also include sugar and pH detectors, or separate test strips for these may be provided.
Particular embodiments are provided as kits, such as field test kits that are readily employed in the field, for instance at the site of a suspected biowarfare agent or bioterrorism contamination. A positive control may also be provided in the kits, to establish that reagent(s) in the kit are functioning properly and/or that the user is following the correct procedures.
An advantage of certain embodiments is that they provide orderly systems for testing suspicious samples that allow obviously safe samples to be eliminated before sophisticated, expensive testing is involved in the analysis. It is particularly envisioned, for instance, that provided methods and kits can be used to assist in triaging possible biowarfare contamination sites and incidents, thereby permitting appropriate allocation of resources.
Another advantage of certain embodiments is that the methods can be used to identify the presence of any pathogen or toxin that includes at least one protein or peptide species, and are not limited to the detection of a particular biological agent as is seen with assays that utilize analyte-specific reagents.
In some embodiments, additional testing is carried out to determine which specific biological agent(s) is present in the sample.
A further advantage of certain embodiments disclosed herein is that protein carrier material (i.e., culture medium) can be detected in addition to the pathogen or toxin, or even in the absence of the pathogen or toxin. Carrier material is a support medium necessary for biological growth. Many biowarfare agents will be prepared and dispersed with significant amounts of carrier material present; the presence of this carrier is an additional indicator that a biological material, potentially hazardous, may be present.
These and other features, aspects, and advantages of the present invention will be better understood with regard to the following description, appended claims, and accompanying drawings.